Bridge peptide beh c18

After trypsin digestion, peptide was desalted by Strata‐X SPE column and vacuum‐dried. Peptide was reconstituted in 20 μL 500 mmol/L TEAB and processed according to the manufacturer's protocol for 8‐plex iTRAQ kit. Briefly, one unit of iTRAQ reagent was all added to peptide solution after thawed and dissolved in 50 μL isopropanol. The peptide mixtures were incubated for 2 hours at room temperature, and then pooled and dried by vacuum centrifugation. The dried and labelled peptide was reconstituted with HPLC solution A (2% ACN, pH 10) and then fractionated into fractions by high pH reverse‐phase HPLC using Waters Bridge Peptide BEH C18 (130 Å, 3.5 μm, 4.6 * 250 mm). Briefly, peptides were first separated with a gradient of 2%‐98% acetonitrile in pH 10 at a speed of 0.5 mL/min over 88 minutes into 48 fractions. Then, the peptides were combined into 12 fractions and dried by vacuum centrifugation. The peptide fractions were desalted using ZipTip C18 according to the manufacturer's instructions. Samples were finally dried under vacuum and kept at −20°C until MS analyses were performed.

Dried and labeled peptides were reconstituted with high-performance liquid chromatography (HPLC) solution A [2% acetonitrile (ACN), pH 10] and then fractionated into fractions by high-pH reversed-phase HPLC using Waters Bridge Peptide BEH C18 (130 Å, 3.5 μm, 4.6 mm × 250 mm). Briefly, peptides were first separated with a gradient of 2–98% ACN with pH 10 at a speed of 0.5 ml/min over 88 min into 48 fractions. Then, the peptides were combined into 16 fractions and dried by vacuum centrifugation. Peptide fractions were desalted using ZipTip C18 according to the instructions of the manufacturer. Finally, the samples were dried under vacuum and kept at −20°C until mass spectrometer (MS) analysis could be performed.